The publication by Wenn (Wenn, R. V. (1975) Biochem. J. 145, 281-285) discloses, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances, involving labelling glycopeptides with a fluorescent labelling reagent, e.g. dansylcadaverine, and separating them by electrophoresis on a paper matrix (paper-electrophoresis) after application to that matrix. The fluorescent labelling reagent imparts a charge to the carbohydrates enabling their electrophoretic separation and it also enables the visualisation of the substances after the electrophoresis.
The publication by West (West, M. H. P., et al. (1984) Electrophoresis 3, 133-138) discloses a technique for the electrophoretic separation of charged substances of relatively low molecular weight, such as amino acids and their oligomers, involving applying the amino acids to an electrophoretic gel made of a polymer of acrylamide, that is a polyacrylamide gel, and separating the substances by the application of an electrical potential to the gel. This technique is known as PAGE. The publication by West also discloses that an electrophoretic gel matrix of relatively high concentration of polyacrylamide enables the separation of the amino acid leucine from its dimer, leucylleucine which in turn is separated from the homologous trimer, leucylleucylleucine and so on. In the publication by West the amino acids were rendered detectable by the use of amino acids that comprised radioisotopic atoms in their structures.
The publication of Weitzman et al. (Weitzman, S., et al. (1979) Anal. Biochem. 97,438-439.) discloses that carbohydrate substances of relatively low molecular weight can be separated by electrophoresis in a polyacrylamide gel when an electrophoretic buffer system that contains borate ions is used.
The publication by Poretz and Pieczenik (Poretz, R. D. and Pieczenik, G. (1981) Anal. Biochem. 115, 170-176) discloses the labelling with a fluorescent labelling reagent of glycopeptides and also discloses their separation after electrophoresis in a polyacrylamide gel and their detection by the incorporated fluorescent label.
The publication by Prakash and Vijay (Prakash, C. and Vijay, I. A. (1983) Anal. Biochem. 128, 41-46) discloses the labelling of carbohydrate substances with a fluorescent labelling reagent.
The publication by Towbin et al. (Towbin, H., et al. (1988) Anal. Biochem. 173, 1-9) discloses the labelling of carbohydrate substances with a chromophore.
The publication by Hase et al. (Hase, S., et al. (1978) J. Biochem. 85, 989-994) discloses the electrophoretic separation by paper electrophoresis in two dimensions of carbohydrate substances labelled with the fluorescent labelling reagent aminopyridine.
The publications by Jackson (Jackson P., (1990) Biochem. J, 270, 705-713 and Jackson, P. 1992) Anal. Biochem. 196, 238-244) and international publications No. WO88/10422, No. WO91/05256, No. WO93/02356, and No. WO92/11531, disclose, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances involving applying carbohydrate substances to an electrophoretic gel to cause differential migration of different substances. The carbohydrate substances may be pre-labelled with a fluorescent labelling reagent, for example 8-aminonaphthalene-1,3,6-trisulphonic acid (known as ANTS for brevity) to impart a charge to the substances, thereby to enable electrophoretic separation, and to enable visualisation of the substances after running the gel. Visualisation may be effected by the naked eye but other means such as photography or electronic imaging may also be used.
The techniques described as examples in the patents which are cited above involve the use of negatively charged or uncharged fluorescent labelling reagents. The separations of fluorescently labelled carbohydrate substances that have been described so far show a relatively limited degree of separation of carbohydrate substances a significant proportion of which are not resolved from each other by the electrophoresis in a useful way. In particular this applies to the asparagine linked glycans that have been analysed as shown in the publication by Stack and Sullivan (Stack, R. J., and Sullivan, M. T. (1992) Glycobiology 2, 85-92). The invention described herein is a novel and innovative development that is related to the methods that are described in the papers and patents cited above.